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Soil Biology & Biochemistry 39 (2007) 684–690 www.elsevier.com/locate/soilbio Short communication Bacterial diversity of terra preta and pristine forest from the Western Amazon

Jong-Shik Kima, Gerd Sparovekb, Regina M. Longoc, Wanderley Jose De Meloc, David Crowleya,Ã

aDepartment of Environmental Sciences, University of California, Riverside, CA, USA bDepartment of , ESALQ, University of Sao Paulo, Piracicaba CP 9, CEP 13418-900, Brazil cDepartment of Technology, Universidade Estadual Paulista, Jaboticabal, SP, CEP 14884-900, Brazil

Received 25 May 2006; received in revised form 1 August 2006; accepted 11 August 2006 Available online 18 September 2006

Abstract

The survey presented here describes the bacterial diversity and community structures of a pristine forest soil and an anthropogenic terra preta from the Western Amazon forest using molecular methods to identify the predominant phylogenetic groups. Bacterial community similarities and species diversity in the two were compared using oligonucleotide fingerprint grouping of 16S rRNA gene sequences for 1500 clones (OFRG) and by DNA sequencing. The results showed that both soils had similar bacterial community compositions over a range of phylogenetic distances, among which Acidobacteria were predominant, but that terra preta supported approximately 25% greater species richness. The survey provides the first detailed analysis of the composition and structure of bacterial communities from terra preta using noncultured-based molecular methods. r 2006 Elsevier Ltd. All rights reserved.

Keywords: Bacterial diversity; Forest soils; Microbial ecology; Terra preta

1. Introduction terra preta and pristine forest soil as the basis for studies on the ecology of these forest soils. Terra preta anthrosols in the are To characterize bacterial diversity in the terra preta and rich soils that contain thick, dark colored, surface horizons pristine forest soil, samples were collected from two with a high organic matter content (Lima et al., 2002) and adjacent locations in the Jamari National Forest, of are noted for their exceptional fertility and ability to Rondonia, Brazil (latitude: 81 450 0 S, longitude: 631 270 0 accumulate stable organic . Prior studies suggest W). The soil from the pristine forest was a these soils were formed by pre-Columbian, Amerindians, having a 1 cm A horizon under a layer of moist duff. The B who practiced a ‘‘slash and char’’ (Mann, horizon consisted of a highly oxidized ultisol with an 2002). The ability of terra preta to accumulate stable pH of 3.1 and had negligible organic matter. The terra organic matter has attracted attention as a possible means preta samples were collected at a site two hundred meters for increasing fertility and carbon storage in tropical soils distant where the soil contained a deep, organic matter (Sombroek, 1966; Lehmann et al., 2003), and has provoked rich, sandy loam in the A horizon that was approximately questions regarding the role of in the 1 m thick before transitioning into the underlying clay formation and plant growth promotion characteristics of ultisol. Intact soil cores measuring 10 cm in depth and 8 cm these soils (Thies and Suzuki, 2003). The objective of this in width were collected from the surface horizon below the study was to carry out a survey of the bacterial diversity in organic litter layer using sterile, stainless steel soil coring sleeves. The cores were shipped immediately to the ÃCorresponding author. Tel.: +1 951 827 3785; fax: +1 951 827 3993. University of California, Riverside, USA, where they were E-mail address: [email protected] (D. Crowley). frozen at 80 1C. At the time of analysis, three cores from

0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2006.08.010 ARTICLE IN PRESS J.-S. Kim et al. / & Biochemistry 39 (2007) 684–690 685 each site were combined into one composite sample joining data were conducted based on 1000 samplings to and sieved to pass a 1.0 mm screen. The composite assess the stability of the phylogenetic relationships. samples were analyzed for their chemical and physical Rarefaction curves and estimates of species diversity at characteristics and metal contents (Supplemental different phylogenetic distances were determined using Table 1, online supplementary material) and were subjec- the computer program DOTUR (Schloss et al., 2004). ted to molecular analyses to identify the predomi- Comparisons of the species coverage and overall commu- nant bacteria and to describe the bacterial community nity similarities at different phylogenetic distances were structures at different phylogenetic distances based on determined using the program LIBSHUFF (Singleton comparisons of 16S rRNA gene sequences recovered from et al., 2001). the samples. The analysis of bacterial diversity employed a two step 2. Results and discussion procedure in which the first step was to sort 16S rRNA clone libraries with a method referred to as oligonucleotide The Amazon contains diverse soils of which only a few fingerprinting of ribosomal genes (OFRG) (Valinsky et al., have been characterized with respect to their microbiology. 2002). This was followed by sequencing of random clones To date there has been only one prior survey of Amazon from the taxonomic clusters generated by OFRG to soils that used molecular methods to analyze soil microbial identify the predominant bacteria. The gene sequences diversity in which 100 clones were analyzed (Borneman and were also used to construct phylogenetic trees based on Triplett, 1997). The survey data presented here thus actual sequence data. To produce the 16S rRNA gene clone provide a more comprehensive survey and the first analysis libraries, near full-length (1465 bp) small-subunit rRNA of terra preta using molecular methods. Inspection of the gene sequences were PCR amplified from soil DNA. The taxonomic trees generated by OFRG showed that the two purified DNA was ligated into pGEM-T (Promega) and soils differed with respect to the number of operational transformed into competent Escherichia coli JM109 (Pro- taxonomic units (OTUs), and the number of branches that mega). Libraries of the rRNA genes were represented by represent different taxonomic groups, with terra preta 768 clones for each sample, which were arrayed on having overall greater diversity (Fig. 1). As shown using replicate nylon membranes for hybridization with 33P rarefaction, there was significant separation of OTU DNA end-labeled oligonucleotide probes as described by richness (95% similarity level) as sample size increased Valinsky et al., (2002). Signal intensities with background above 50 for each sample set (Fig. 2). Enumeration of correction were obtained using ImaGene 4.0 software the unique 16S rRNA gene sequences yielded 396 (Biodiscovery) and were transformed for every probe OTUs from terra preta as compared to 291 OTUs in the into three values: 0, 1, and N, where 0 and 1 indicate forest soil (Table 1). Calculation of Shannon index values negative and positive hybridization events, respectively, using DOTUR yielded significantly different (P40:05) and N indicates an uncertain assignment. This process values for terra preta and forest soil (5.2 and 4.37, creates a hybridization fingerprint for each clone, which is respectively). Other commonly used measures of diversity a vector of values resulting from hybridizations with all including Simpsons Index of Diversity and Chao I, which probes. Estimates of diversity using 16S rRNA gene employ independent mathematical approaches to measur- sequences were examined based at 95%, and 90% ing diversity revealed the same phenomenon. Altogether, similarities using the PHYLIP-formatted distance matrices these analyses suggest that bacterial species richness was generated by the computer program PAUP 4.0 (Sinauer approximately 25% greater for terra preta than that in the Associates, Inc.). forest soil. To identify the taxonomic groups separated by OFRG, Using the program LIBSHUFF to compare the simila- nucleotide sequences were determined for 76 and 49 rRNA rities of the communities from each soil at different gene clones that were randomly selected from within each phylogenetic distances, it was determined that overall of the clusters generated by OFRG. The 16S rRNA gene coverage of sequence diversity was high, with values of sequences were submitted to the BLAST server (Basic 60% and 68% for the forest soil and terra preta samples Local Alignment Search Tool), National Center for sets, respectively (Fig. 3). Comparison of the forest soil Biotechnology Information (NCBI) to determine the sample set against terra preta revealed that all of the closest matching sequences in the GenBank and to infer OTU diversity in the forest soil was represented in terra phylogenetic affiliations. Novel sequences were deposited preta and that the forest sample was not signifi- at GenBank and were assigned accession numbers cantly different from the terra preta (P ¼ 0:64; data not AY326512–AY326636. shown). In contrast, comparison of the terra preta versus Phylogenetic trees based on the sequenced clones were the forest soil showed that terra preta was significantly constructed using the 16S rRNA gene sequence data. different (P ¼ 0:05), in that it contained additional Nucleotide sequences were aligned using Clustal X, after sequences that did not occur in the forest soil. The greatest which an evolutionary distance matrix was generated difference in 16S rRNA gene sequences between terra using the program MEGA3 (Kumar et al., 2004) using preta and the forest soil occurred for OTUs having up to the N-J method. Bootstrap analyses of the neighbor- 5% evolutionary distance (95% 16S rRNA similarity), ARTICLE IN PRESS 686 J.-S. Kim et al. / Soil Biology & Biochemistry 39 (2007) 684–690

Terra Preta Preserved Forest Nitrospira Proteobacteria Chloroflexi Acidobacterium Bacillus Proteobacteria

Acidobacterium

Acidobacterium

alpha-Pro

Actinobacteria

CFB gamma-Pro

beta-Pro Verrucomicrobia Acidobacterium

Actinobacteria

alpha-Pro

Verrucomicrobia Actinobacteria Delta-Pro Chloroflexi gamma-Pro beta-Pro Planctomycetes Proteobacteria Acidobacterium

Verrucomicrobia Actinobacteria 0.05

Fig. 1. Taxonomic cluster analysis of 16S rRNA gene sequences from terra preta and adjacent pristine forest soil based on oligonucleotide fingerprinting of 16S rRNA gene sequences. The 16S rRNA clone libraries were generated for single samples consisting of three composited replicate soil cores (litter layer removed) taken from the top 10 cm of the soil profile at each location.

corresponding to species and genera. The coverage curves cetes, and Verrucomicrobia. Representatives of the a, b, converged at 95% evolutionary distance and then sepa- and g Proteobacteria were similarly abundant in terra rated again at distances from 96% to 90%, indicating that preta; whereas mostly a Proteobacteria were found in the there were also significant differences between the commu- forest soil. nities at deeper phylogenetic levels. Complete coverage of Acidobacterium sp are common in many forest soils the two communities was obtained at greater than 90% around the world, comprising from 12% of the non- similarity, suggesting that the large data set that was cultured species surveyed in Austrian forest soils under analyzed here, with ca. 700 clones per sample, represented pine to 35% in spruce-fir-beech soils in Europe (Hackl all of the major bacterial phyla that were present in the et al., 2004). At least four subgroups of these bacteria have soils. been recognized previously using specific 16S rRNA gene The differences in community composition revealed by primers in a survey of 43 soils and sediments representing a the LIBSHUFF analysis were confirmed by the phyloge- range of environments (Barns et al., 1999). This earlier netic analyses based on DNA sequencing. Identities of the survey suggested that the A and G subgroups are major bacterial groups by direct sequencing of clones ubiquitous, whereas the Y and O subgroups are largely representing different clusters revealed 14 phylogenetic absent from acid soils. The A subgroup is thought to be the groups in terra preta, as compared to 9 from the forest soil most phylogenetically diverse, with deep branches in the (Fig. 4). Among the major groups represented, Acidobac- phylogenetic tree that may represent distinct lineages. Our terium was predominant, comprising approximately 30% results are largely in agreement with these suppositions. of the bacteria in terra preta and 50% of the forest soil. Here, most of the cloned bacterial sequences were in the The phylogenetic trees included two possible new clades A and G subgroups. Terra preta contained sequences from of Acidobacterium that were distinct from the pre- the Y subgroup, which purportedly favors non-acid soils. viously described subgroups for this phylum. Other Only the A subgroup was found in the acid soil from the bacterial groups that were common to both two soils pristine forest. In the phylogenetic tree generated by DNA included the Proteobacteria, Actinobacteria, Planctomy- sequencing, the BLAST analysis revealed three possible ARTICLE IN PRESS J.-S. Kim et al. / Soil Biology & Biochemistry 39 (2007) 684–690 687

350

Terra Preta 300 Forest Soil

250

200

150

Number of OTUs observed 100

50

0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 Number of sequences sampled

Fig. 2. Rarefaction analysis of 16S rRNA gene sequences from terra preta and forest soil. Gene sequences were differentiated by oligonucleotide fingerprint grouping at 0.05 dissimilarity level. Vertical bars indicate 95% confidence intervals.

Table 1 1.00 Measures of bacterial species diversity based on OFRG analysis of 0.0030 microbial communities from terra preta and Amazon forest soil 0.80 0.0025 Index Terra preta Forest soil 2 0.60 0.0020 OTU richness 0.0015

0.40 (Cx-Cxy)

Unique OFRG groups / clones 396/742 291/768 Coverage, C 0.0010 0.05 evolutionary distance 287 196 0.20 0.10 evolutionary distance 210 142 0.0005 Shannon index 5.2 4.37 0.00 Simpsons index 0.015 0.34 0.0 0.1 0.2 0.3 0.4 0.5 Estimated richness Chao1 (0.05 413 277 evolutionary distance) Evolutionary distance, D Fig. 3. LIBSHUFF analysis of 16S rRNA gene sequences from terra preta (closed circles) compared to adjacent forest soil (open circles) showing differences in composition of the bacterial communities (y-axis) over a range of evolutionary distances (D)(x-axis). Solid lines indicate the value new subgroups of Acidobacterium. Two new clades of of (CX-CXY)2 for samples at each value of D. Broken lines indicate the Acidobacterium were found in the terra preta, one that was P ¼ 0:05 value of (CX-CXY)2 for the randomized samples. most closely related to the G subgroup, and a second well- defined cluster of five clones that were most closely related to the Y-subgroup. The new clade of Acidobacterium in the forest soil was comprised by four clones that occurred composition of forest soils is still not understood, but likely within the A-subgroup and were most similar to previous contributes to differences in diversity and community accessions designated as Holophaga. composition along the forest floor. There was also Based on prior knowledge, soil pH is likely to be one of increased activity and soil aggregation in terra the most important selection factors affecting the microbial preta that may provide a wide range of niches and thereby species composition in different soils (Fierer and Jackson, contribute to increased bacterial species diversity. Addi- 2006). Here, both soils supported abundant plant tional surveys and comparisons at different locations will growth and carried similar above ground vegetation be needed to characterize other locations with terra preta. consisting of a diverse community of Amazon forest It may then be possible to unravel the ecology of these tree species and understory plants. The relationship bacterial communities and study the role of specific between organic matter inputs from different overstory bacterial groups that contribute to the many interesting trees, effects, and the bacterial community properties of these soils. ARTICLE IN PRESS 688 J.-S. Kim et al. / Soil Biology & Biochemistry 39 (2007) 684–690

91 869-1 Uncultured Acidobacteria (95%) 99 22-1 Uncultured Acidobacteria ( 95%) 91 26-1 Uncultured Acidobacteria (94%) G 73 99-1 Uncultured Acidobacteria (96%) 210-1 Uncultured Acidobacteria (92%) 99 79 1192-1 Uncultured Acidobacteria (95%) 997-1 Uncultured Acidobacteria (92%) 99 483-1 Uncultured Acidobacteria (87%) New 99 894-1 Uncultured Acidobacteria (93%) 291-1 Uncultured Acidobacteria (95%) 1086-1 Uncultured Acidobacteria (92%) Acidobacterium 18-1 Uncultured Acidobacteria (92%) 83 991-1 Uncultured Acidobacteria (97%) 99 1271-1 Uncultured Acidobacteria (94%) A 97 1272-1 Uncultured Acidobacteria (94%) 9999 1005-1 Uncultured Acidobacteria (93%) 99 1091-1 Uncultured Acidobacteria (97%) Y 70 395-1 Uncultured Acidobacteria (93%) 1093-1 Uncultured Acidobacteria (91%) 99 1267-1 Uncultured Acidobacteria (92%) 99 1363-1 Uncultured Acidobacteria (93%) New 99 7-1 Uncultured Acidobacteria (93%) 87 501-1 Uncultured Acidobacteria (91%) 81 31-1 Uncultured Acidobacteria (93%) 40-1 Streptomyces kasugaensis (94%) 99 37-1 Streptomyces kasugaensis (94%) 34-1 Streptomyces kasugaensis (94%) 99 42-1 Streptomyces kasugaensis (94%) Actinobacteria 41-1 Streptomyces kasugaensis (94%) 75 911-1 Uncultured Rubrobacteridae (93%) 15-1 Uncultured Rubrobacteridae (95%) 99 43-1 Paenibacillus macerans (95%) 99 1078-1 Bacillus cereus (98%) 99 1077-1 Bacillus cereus (99%) Bacillus 993-1 Bacillus bataviensis (96%) 99 307-1 Bacillus bataviensis (97%) 98 1180-1 Uncultured Chloroflexi (87%) 1067-1 Uncultured Chloroflexi (92%) Chloroflexi 99 12-1 Uncultured Chloroflexi (94%) 98 99 4-1 Parachlamydia acanthamoebae (93%) Chlamidiae 3-1 Parachlamydia acanthamoebae (94%) 99 27-1 Uncultured Verrucomicrobia (95%) 28-1 Uncultured Verrucomicrobia (95%) Verrucomicrobia 30-1 Uncultured Verrucomicrobia (95%) 1164-1 Nitrospira moscoviensis (96%) 1163-1 Nitrospira moscoviensis (95%) Nitrospira 99 6-1 Uncultured Bacteroidetes (92%) 5-1 Uncultured Bacteroidetes (92%) Bacteroidetes 99140-1 candidate division TM7 (89%) TM7 227-1 Afipia genosp (93%) 99 99 128-1 Afipia genosp (93%) 98 594-1 Uncultured alpha proteobacterium (97%) Alpha Proteobacteria 99 711-1 Bradyrhizobium japonicum (98%) 1100-1 Bradyrhizobium japonicum (98%) 597-1 Mesorhizobium sp. (90%) 54 776-1 Uncultured gamma proteobacterium (92%) Gamma Proteobacteria 82 47-1 Nitrosococcus halophilus (90%) 99 1083-1 Uncultured beta proteobacterium (95%) 99 966-1 Uncultured beta proteobacterium (89%) 1251-1 Burkholderia sp. (98%) 99 99 592-1 Uncultured beta proteobacterium (94%) Beta Proteobacteria 78 591-1 Uncultured beta proteobacterium (94%) 142-1 Burkholderia sp. (98%) 141-1 Burkholderia sp. (98%) 2-1 Unknown 46-1 Uncultured Planctomycete (93%) Planctomycetes 99 426-1 Anaeromyxobacter dehalogenans (90%) 293-1 Uncultured delta proteobacterium (92%) 89 685-1 Geobacter sulfurreducens (87%) 99 1074-1 Geobacter sulfurreducens (87%) 24-1 Syntrophus gentianae (88%) Delta Proteobacteria 900-1 Uncultured delta proteobacterium (92%) 221-1 Uncultured delta proteobacterium (95%) 94 772-1 Uncultured delta proteobacterium (95%) 99 1154-1 Uncultured delta proteobacterium (96%) 1001-1 Uncultured delta proteobacterium (96%) 98

0.05 (A)

Fig. 4. Phylogenetic trees for unique 16S rRNA gene sequences based on rRNA gene sequencing. (A) Terra preta soil (B) Forest soil. ARTICLE IN PRESS J.-S. Kim et al. / Soil Biology & Biochemistry 39 (2007) 684–690 689

98 340-2 Uncultured Acidobacteria (92%) 355-2 Uncultured Acidobacteria (91%) 100 339-2 Uncultured Acidobacteria (92%) 753-2 Uncultured Acidobacteria (92%) 100 169-2 Uncultured Acidobacteria (92%) 1209-2 Uncultured Acidobacteria (92%) 1496-2 Uncultured Acidobacteria (92%) 466-2 Uncultured Acidobacteria (93%) A 1151-2 Uncultured Acidobacteria (93%) 1215-2 Uncultured Acidobacteria (92%) 100 1216-2 Uncultured Acidobacteria (92%) 81-2 Uncultured Acidobacteria (94%) Acidobacteria 92-2 Uncultured Acidobacteria (94%) 100 100 1031-2 Uncultured Acidobacteria (95%) 1219-2 Uncultured Acidobacteria (88%) 244-2 Uncultured Acidobacteria (96%) 83 821-2 Uncultured Acidobacteria (94%) 760-2 Uncultured Acidobacteria (95%) New 99 100 99 958-2 Uncultured Acidobacteria (94%) 829-2 Uncultured Acidobacteria (93%) 648-2 Uncultured Acidobacteria (94%) 462-2 Uncultured Acidobacteria (92%) A 100 55-2 Uncultured Acidobacteria (96%) 100 100 157-2 Uncultured Acidobacteria (96%) 90-2 Frateuria aurantia (96%) 100 845-2 Uncultured gamma proteobacteria (96%) Gamma Proteobacteria 96 1052-2 Frateuria aurantia (96%) 99 1048-2 Burkholderia unamae (97%) Beta Proteobacteria 100 1131-2 Burkholderia tropica (98%) 1236-2 Uncultured gamma proteobacterium (94%) 100 52-2 Uncultured alpha proteobacterium (97%) 71 557-2 Bradyrhizobium genosp (99%) 99 98 722-2 Sphingomonas sp. (96%) 670-2 Afipia felis (89%) 100 178-2 Uncultured alpha proteobacterium (95%) Alpha Proteobacteria 100 288-2 Azospirillum amazonense (92%) 87 337-2 Acidosphaera rubrifacien (95%) 1503-2 Acidosphaera rubrifacien (91%) 100 100 1529-2 Acidosphaera rubrifacien (93%) 1202-2 Uncultured actinobacterium (92%) 1309-2 Acidothermus cellulolyticus (92%) 100 Actinobacteria 100 1532-2 Frankia sp. (92%) 271-2 Unknown 100 1126-2 Uncultured planctomycete (90%) 345-2 Nostocoida limicola (96%) Planctomycetes 530-2 Parachlamydia acanthamoebae (93%) Chlamydiae 546-2 Uncultured Verrucomicrobia (96%) 241-2 Uncultured Verrucomicrobia (89%) 100 Verrucomicrobia 100 941-2 Uncultured Verrucomicrobia (88%)

0.05 (B)

Fig. 4. (Continued)

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